It was postulated by us that melanin biosynthesis cannot take place unless the melanosomal pH is acidic. That melanosomal pH may be acidic was suggested by other researchers as well. However, the actual pH was never determined. The current study was undertaken to estimate the intramelanosomal pH. Lightly melanized and heavily melanized melanosomes from B16 murine melanoma tissue were prepared by sucrose density gradient centrifugation and their internal pH was estimated. It was shown that quenching of fluorescence of 9-aminoacridine by melanosomes was to a large extent dependent on the pH difference between melanosomes and the medium in which they were suspended and it was used to determine the pH of lightly and heavily melanized melanosomes from B16 melanoma. The pH of lightly melanized melanosomes and heavily melanized melanosomes was found to be in the range of 4.2 to 4.6 and 3.0, respectively. These organelles were shown to hydrolyze fluorescein diacetate to fluorescein. The excitation spectrum of fluorescein generated in lightly melanized melanosomes and heavily melanized melanosomes also indicated that the pH in lightly melanized melanosomes was 4.6 and lower than 4.6 in heavily melanized melanosomes. The enzymes are active at such low pH. These organelles are also shown to contain proton-translocating ATPase, which may be involved in the acidification of lightly melanized melanosomes, possibly in addition to melanin biosynthesis. These results confirm our original postulate that melanin biosynthesis that occurs in melanosomes takes place under acidic conditions.